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In-Depth Information
but require appropriate precautions during
sample preparation.
26,27
Concerted effort to iden-
tify which metabolites are and are not affected by
different quenching and/or storage conditions is
extremely important as the
chemical shifts inNMR, so control of these param-
eters is crucial for optimal results, with buffering
to 0.3 M recommended.
33
Recent comparison of the simple dilute-and-
shoot strategy with the liquid
e
liquid extraction
(LLE) method showed higher intensity and higher
number of features, with the LLE method possibly
presenting a new avenue to further improve
metabolite coverage of urine samples.
30
The deple-
tionof ureausingurease is currentlya controversial
sample pretreatment step in global metabolomics
of urine. This sample pretreatment step involves
incubation with 30 to 100 units of urease enzyme
for complete urea removal, followed by precipita-
tion with methanol or ethanol to remove the
enzyme.
34
It successfully removes urea interfer-
ence from chromatography (both LC-MS and
GC-MS); however, serious alteration of metabolite
pro
field of metabolomics
matures and moves forward in successful
biomarker discovery. Once such data is available,
the question of whether metabolism quenching
step is truly necessary in global metabolomics
can be answered in a more holistic manner.
SAMPLE PREPARATION
METHODS FOR BIOFLUIDS
Dilute-and-Shoot: Preferred Method for
Urine Metabolomics
The most commonly employed method for
global metabolomics of urine is the
les in combination with urease treatment was
also reportedwith signi
“
dilute-and-
cant decreases inmetabo-
lite levels such as acotinic acid, hypoxanthine, aco-
nitate, citrate, succinate, ascorbate, tyrosine, and
glycerol.
37
Figure 3
summarizes the recommended
LC-MS protocol for urine and other biological
shoot
strategy, and a detailed protocol for urine
metabolomics is available.
28
”
Typical dilution
factors with puri
ed water range between 1:1
and 1:10 prior to LC-MS analysis.
28
e
30
Neat
samples can also be analyzed to enhance the
detection of low abundance metabolites,
provided that adequate column washing regime
is employed.
31,32
Sodium azide (0.1%) is some-
times added to prevent bacterial growth during
storage,
28
fluids discussed in subsequent sections.
Solvent Precipitation: Preferred Method
for Plasma, Serum, and Other Bio
uids
The most commonly employed method for
global metabolomics of blood and cerebrospinal
filtration was recently
found to be superior for bacterial removal.
23
No
preservative addition is necessary in combination
with NMR and/or LC-MS analysis if samples
are sterile and stored at or below
e
20
C.
32
e
35
Mild precentrifugation (1,000
e
3,000 rcf [relative
centrifugal force] for 5 min) immediately after
sample collection is recommended
23
in order to
effectively remove cellular components while
minimizing the cell breakage that can occur with
high centrifugation speeds.
31
Considering the
large diurnal variability of urine, 24-hour collec-
tionperiods andurine storage on ice orwith refrig-
eration is recommended for both animal and
human studies.
36
Finally, the normal variability
of pH and ionic strength of urine can cause small
but 0.20
m
m
fluid (CSF) is protein precipitation with organic
solvent (methanol, acetonitrile, ethanol, acetone,
or combination).
9,13,38
e
40
In addition to protein
removal, the addition of organic solvent to a bio-
fluid sample also disrupts any binding between
metabolite with proteins present in solution,
and thus the metabolite concentrations obtained
represent total metabolite concentration, which
is equivalent to the sum of bound and unbound
(free) metabolite concentrations. Solvent precipi-
tation is the preferred method in metabolomics
because it is the most unselective and thus
provides high metabolite coverage. However,
the serious consequences of poor sample cleanup
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